Project Summary: Xerostomia, or ?dry mouth?, is a challenging clinical condition, caused by damage to the cells of the salivary gland. It may result from a variety of tissue insults, including acute damage from radiation therapy for head and neck cancers, progressive auto-immune response in Sjogren?s disease, or other unknown etiology from aging. Current treatments offer only temporary relief of symptoms, and poor resolution of associated oral health decay. The cost of this condition is considerable, both in quality of life and the financial burden of increased dental care. The fields of tissue engineering and regenerative medicine offer many tools for the potential reconstitution of healthy salivary-derived cells within supportive hydrogel matrices, but few of these options provide sufficient spatial and temporal resolution to restore the complex branched structure and precise spatial phenotype map of the major salivary glands. However, new discoveries in laser-based hydrogel degradation (LBHD) can be used to ?carve? pathways through intact hydrogel slabs, with pinpoint, subcellular resolution in xyz, and offer a method to guide a growing salivary epithelial bud in 3 dimensions. Our hypothesis for the present proposal is that we can use multiphoton-based LBHD to elongate a multicellular cluster in a given direction, and recreate key elements of the native gland. To do this, we will employ our laboratory?s expertise in isolation of primary human salivary-derived stem/progenitor cells (hS/PCs) from healthy tissues, and encapsulation as responsive 3D multicellular spheroid clusters within customizable, biocompatible hyaluronic acid (HA) hydrogels. Our ongoing work has shown that, by tailoring the porosity of these hydrogels and their concentration of bioactive epitopes, we can impact cluster size, morphology, and interaction with the surrounding extracellular matrix. We will test our hypothesis through the following Specific Aims: Aim 1. Establish parameters to carve ?tunnels? through HA hydrogels and promote HS/PC cluster ingrowth. Aim 2. Adapt the system to alternate matrices that enable fibroblast co-culture, or incorporate photolabile crosslinkers for easier fabrication. Aim 3. Assess phenotype of the growing cluster, at its trailing and leading edges and branched termini, for signs of differentiated phenotype. If successful, this system could serve as a useful model for studying mechanisms of human salivary cell organization and differentiation; the system might also be an early prototype for manufacturing tissue engineered gland replacements. The R03 mechanism will provide support for the necessary pilot and feasibility studies, to demonstrate that these proven technologies can be combined to produce a novel platform.